[Background] Enterotoxigenic Escherichia coli K99 is one of the main pathogens causing diarrhea in piglets. So far the treatment of this disease mainly depends on antibiotics, but the long-term use of antibiotics has caused the increase of drug resistance, therefore, new product is urgently needed to solve this problem. [Objective] A lytic phage against Enterotoxigenic Escherichia coli K99 was isolated from the manure of livestock and identified, and its biological properties were assayed as well. The preventive and therapeutic effects of the bacteriophage on Enterotoxigenic Escherichia coli K99 infection were examined in mice. [Methods] The lytic phage was isolated by the double-layer agar culture method and the phage plaque was checked. Morphology of the phage was observed by transmission electron microscopy and the type of nucleic acids was also identified after the phage purification. The biological parameters including the thermal stability, pH stability and optimal multiplicity of infection (MOI) were assayed separately. The inhibitory effects of the bacteriophage on Escherichia coli infection in mice was tested by in vivo experiments with mice. [Results] A lytic phage (designated as ФK99-1) against Enterotoxigenic Escherichia coli K99 was successfully isolated from livestock manure. ФK99-1 belongs to family Myoviridae according to the electron microscopy and type identification of nucleic acid. The phage could withstand the temperature up to 50 °C and maintain stable titer under pH 3.0–10.0. The optimal MOI of ФK99-1 was 0.000 01, and the burst size was 108 333 PFU/cell. Application of ФK99-1 in mice which were infected with Enterotoxigenic Escherichia coli K99 could alleviate the related symptoms and viscera lesions. [Conclusion] The isolated phage, ФK99-1, belongs to lytic phage of Myoviridae, which shows strong adapting capacity to various temperature and acid-base environments. At the same time the isolated phage shows inhibitory effect on E. coli infection in mice.